Individual patient-dependent influence of erythrocyte lysing procedures on flow-cytometric analysis of leukocyte subpopulations

Background: It is known from the literature that the use of erythrocyte lysing reagents in flow-cytometric analysis results in a significant loss of immunolabelled cells, e.g. T helper and T supressor lymphocytes (CD4+/CD8+ T cells) or stem cells (CD34+ cells). Although WBC subset-specific variations in the sensitivity to lysing reagents are already described, the same variations are possible for each individual. In the present study we investigate the individual and patient-dependent loss of CD4+ T cells in blood samples by the use of a true volumetric flow cytometer (TVC) and a new no-lyse no-wash procedure. Material and Methods: Blood of 50 individuals was used for flow-cytometric counting of CD4+ T cells, and cells were counted by the use of a lyse no-wash and a no-lyse no-wash protocol (600 single measurements). The reproducibility of the loss of CD4+ T cells was measured by 10 individually prepared samples out of a single blood sample; each sample was measured 3 times (n = 30). Results: The mean lysing-dependent CD4+ T-cell loss out of the sample from a single individual was 15% (range 12-18%). The reproducibility of the determination from the same individual was high (coefficient of variation (CV) = 12%). Measuring the concentration of CD4+ T cells in blood samples of different individuals, the mean loss of CD4+ T cells was 9.8% (range 0-23%), interestingly with a high variability and a CV of 65% In = 600). Therefore, most current flow-cytometric protocols with erythrocyte lysing procedure do not allow reproducible and accurate determinations of CD4+ T cells because of individual leukocyte losses. Conclusion: Lysing-dependent cell loss does not represent a constant or calculable parameter in leukocyte subclass enumeration. Beside the leukocyte subset-specific differences in sensitivity to erythrocyte lysing reagents, patient disease and drug treatment may influence the stability of leukocytes under investigation. Accordingly, the present study shows large lysing-dependent interindividual differences in counting results of CD4+ T cells.